REVERSE GENOMICS OF REGULATORY ELEMENTS GOVERNING SPLICING

描述(申请人提供)：人类个体基因组的差异导致了人类的大部分多样性。人类基因组计划已经过去了十年，但基因组如何控制表型仍然是一个谜。像ENCODE这样的联盟试图通过使用全基因组数据集将功能输出与序列相关联来识别人类和其他模式生物中的功能DNA元件。然而，这些研究并不一定会提高我们解释DNA元素在新的背景下或突变时的作用的能力。这样的理解对于预测序列改变对表型的影响以及为未来的医学或技术目的而设计生物学是至关重要的。DNA元件的组合作为密码控制特定的功能，如转录、剪接、定位和沉默。破译这些编码是困难的，因为有限的自然变异集通常不足以控制变量，如序列组成或元素组合。要证明特定序列对基因表达有因果影响，需要进行仔细控制的反向遗传学研究。在全基因组范围内进行这样的实验是困难的，因为我们无法(1)迅速改变单个遗传元素的序列和背景，(2)量化数千种这样的改变的后果。我的中心愿景是破译控制基因表达的顺式调控密码，方法是使用已定义的合成DNA文库的多路测量，将反向遗传学实验扩展到基因组规模。我将在我工作的基础上，开发下一代基因合成技术和多路报告分析，通过同时进行数千项受控实验测试，系统地确定控制哺乳动物基因表达的序列是如何协同作用的。在这里，我们将应用这些技术发展来研究基因调控元件如何控制前mRNA剪接过程。控制剪接的主要序列元件，即剪接供体、受体和分支位点，不能传递足够的信息来单独指定外显子的包含或排除。其他调控元件，如外显子或内含子剪接增强子和抑制子，已知影响复杂密码中的剪接，该密码可以根据组织或细胞类型的不同而变化。我们将利用这里提出的新技术发展来系统地询问和提炼剪接代码。研究剪接将有助于集中开发一整套工具和技术，这些工具和技术稍后将使我们能够攻击控制基因调控的其他形式的顺式元件。

DESCRIPTION (provided by applicant): Differences in our individual genomes give rise to most of human diversity. A decade removed from the Human Genome Project, much of how the genome directs phenotype still remains a mystery. Consortiums like ENCODE seek to identify functional DNA elements in humans and other model organisms by correlating functional outputs with sequence using genome-wide data sets. However, these studies do not necessarily improve our ability to interpret how DNA elements act in new contexts or when mutated. Such an understanding will be critical to predict the effects of sequence alterations on phenotype and to engineer biology for future medicinal or technological purposes. Combinations of DNA elements act as codes controlling particular functions like transcription, splicing, localization, and silencing. Deciphering these codes is difficult, as the limited set of natural variants is typically insufficient to control for variables such as sequence composition or element combinations. Proving that particular sequences have causative effects on gene expression requires carefully controlled reverse genetic studies. Conducting such experiments on genome-wide scales is difficult because of our inability to (1) rapidly alter the sequence and context of individual genetic elements and (2) quantify the consequences of thousands of such changes. My central vision is to decipher cis-regulatory codes controlling gene expression by scaling reverse genetics experiments to genomic scales using multiplexed measurements of defined synthetic DNA libraries. I will build upon my work developing next-generation gene synthesis technologies and multiplexed reporter assays to systematically determine how sequences governing mammalian gene expression act in concert by doing thousands of controlled experimental tests simultaneously. Here, we will apply these technological developments to study how genetic regulatory elements control the process of pre-mRNA splicing. The major sequence elements controlling splicing, namely the splice donor, acceptors, and branch sites, do not convey enough information to specify exon inclusion or exclusion alone. Other regulatory elements, such as exonic or intronic splicing enhancers and suppressors, are known to affect splicing in a complex code that can vary based on tissue or cell type. We will systematically interrogate and refine the splicing code by leveraging the new technological developments proposed here. Studying splicing will help focus development of a complete suite of tools and technologies, which will later let us attack other forms of cis-elements controlling gene regulation.